The present invention provides an N-terminally truncated HER-2/neu polypeptide, p95HER-2, that is useful as a diagnostic and prognostic indicator for breast cancer. The present invention further provides a 12-15 amino acid xe2x80x9cextracellular stubxe2x80x9d polypeptide that is also a useful epitope for an immunological assay for diagnosis and prognosis of various adenocarcinomas, particularly breast cancer and ovarian cancer.
The present invention was made with funding from the United States Government under grant CA-71447 from the National Cancer Institute and DAMD17-6204 from the Department of Defense (DOD) Breast Cancer Research Program. The United States Government may have certain rights in this invention.
The HER-2/neu (erbB-2) gene encodes a receptor-like tyrosine kinase (RTK) which is a member of the epidermal growth factor receptor family (Coussens et al., Science 230:1132-1139, 1985). Overexpression of HER-2/neu has been observed in tumors arising at many sites including non-small cell lung (Kern et al., Cancer Res. 50:5184-5191, 1990), colon (Cohen et al., Oncogene, 4:81-88, 1989), prostate (Arai et al., Prostate 30:195-201, 1997), ovarian, and breast (Slamon et al., Science 244: 707-712, 1989). In human breast cancer, where HER-2/neu involvement has been studied, overexpression occurs in 15-30% of the cases (Singleton and Strickler, Pathol.Annual 27 Pt 1:165-198, 1992) and predicts for significantly lower survival rate and shorter time to relapse in patients with lymph node positive disease (Slamon et al., Science 244: 707-712, 1989; Singleton and Strickler, Pathol.Annual 27 Pt 1:165-198, 1992; Slamon et al., Science 235:177-182, 1987; and Slamon et al., Science 235:177-182, 1987). The significance of HER-2/neu in node negative patients is controversial and so far its clinical utility as a prognostic indicator is limited (Slamon et al., Science 235:177-182, 1987; and Hynes et al., Biochem. Biophys. Acta 1198:165-184, 1994). Various approaches are being taken toward HER-2/neu targeted therapeutics many of which are based on antibodies specific to the extracellular domain (ECD) of the transmembrane protein, which either down regulate receptor function or target recombinant toxins with the goal of specifically killing HER-2/neu expressing tumor cells (Hynes et al., Biochem. Biophys. Acta 1198:165-184, 1994; Press et al., Progress in Clinical and Biological Research 354: 209-221, 1990; and Dougall et al., Oncogene 9: 2109-2123, 1994).
In addition to the full length transmembrane product, p185, of the HER-2/neu gene, a truncated product corresponding to the extracellular domain (ECD) is released from breast carcinoma cells in culture by regulated proteolysis (Lin and Clinton, Oncogene 6:639-643, 1991; Zabrecky et al., J. Biol. Chem. 266: 1716-1720, 1991; and Pupa et al., Oncogene, 8:2917-2923, 1993), and is also produced from an alternative transcript (Scott et al., Mol. Cell. Biol., 13:2247-2257 1993). HER-2/neu ECD is elevated in the serum of patients with breast (Leitzel et al., J. Clin. Oncol. 10:1436-1443, 1992), ovarian (Maden et al., Anticancer Res. 17:757-760, 1997), and prostate cancer (Myers et al., Int. J. Cancer 69 398-402, 1996). Several studies of breast cancers estimate that 6% or less of early stage breast cancer, about 25% of patients with metastatic and locally advanced disease, and greater than 50% of patients with recurrent metastatic disease have elevated serum ECD (Brandt-Rauf et al., Mutation Res. 333:203-208, 1995). Elevated ECD, in serum is associated with overexpression of HER-2/neu in tumor tissue and also has been correlated to tumor load (Molina et al., Br. J. of Cancer 4:1126-1131, 1996; and Brodowicz et al., Oncology 54:475-481, 1997). Serum ECD is a marker of metastatic disease and may predict recurrence (Molina et al., Br. J. of Cancer 4:1126-1131, 1996), shortened survival (Brodowicz et al., Oncology 54:475-481, 1997; Kandl et al., Br. J. Cancer 70:739-742, 1994; Fehm et al., Oncology 55:33-38, 1998 and Mansour et al., Anticancer Res. 17:3101-3105, 1997), and response to antiestrogen therapy in advanced stage patients (Leitzel et al., J. Clin. Oncol. 13:1129-1135, 1995; and Yamauchi et al., J. Clin. Oncol. 15:2518-2525, 1997). Serum ECD has also been reported to neutralize the activity of anti HER-2/neu antibodies targeted to the ECD (Baselga et al., J. Clin. Oncol. 14:737-744, 1996; and Brodowicz et al., Int. J. Cancer. 73:875-879, 1997) possibly allowing escape of HER-2-rich tumors from immunological control.
Cellular fragments created by ectodomain shedding have been described for the colony stimulating factor receptor (CSF-1R) (Downing et al., Mol. Cell. Biol. 9:2890-2896, 1989), the TrkA neurotrophin receptor (Cabrera et al., J. Cell. Biol. 132 427-436, 1996), Axl receptor (O""Bryan et al., J. Biol. Chem., 270.551-557, 1995), and HER-4 (Vecchi et al., J. Biol. Chem. 271:18989-18995, 1996). However, a truncated cellular product of HER-2/neu shedding has not been identified. The truncated CSF-1R was found to have in vitro kinase activity (Downing et al., Mol. Cell. Biol. 9:2890-2896, 1989), and the cytoplasmic HER-4, induced by phorbol ester tumor promoters, had little or no kinase activity (Vecchi et al., J. Biol. Chem. 271:18989-18995, 1996) while a truncated HER-4 found in cells treated with a proteosome inhibitors was an active kinase (Vecchi et al., J. Cell. Biol. 139:995-1003, 1997). Therefore, there is a need in the art to identify a truncated HER-2/neu polypeptide and determine if it has enzymatic activity in general or kinase activity in particular. Moreover, such a truncated polypeptide is likely to be a better marker for tumor diagnosis, screening and prognosis as it will be easier to assay for the polypeptide than to assay for shed ECD, which is present in a much more dilute form.
The ECD of full-length transmembrane receptors often exerts a negative regulatory constraint on their signaling activity. Engineered deletion of a region of the HER-2 ECD was found to enhance its oncogenic potency (DiFiore et al., Science 237:178-182, 1987; Hudziak et al., Proc. Natl. Acad. Sci. USA 84: 7159-7163, 1987; Segatto et al., Mol. Cell. Biol. 8:5570-5574, 1988; and Bargmann and Weinberg, EMBO J. 7:2043-2052, 1988). This has also been illustrated by engineered removal of the ECD from the epidermal growth factor (EGF) receptor and by the oncogenic potency of viral encoded v-erbB, v-kit, and v-ros, that are missing regions of the ECD found in their normal cellular counterparts (Rodrigues and Park, Curr. Opin. Genet. Dev. 4:15-24, 1994). Naturally occurring mutant EGF receptors with N-terminal truncations have been identified in several human carcinomas (Moscatello et al., Cancer Res., 55:5536-5539, 1995) and have constitutive signaling activity and enhanced oncogenic transforming activity in cell culture and animal models (Moscatello et al., Oncogene 13:85-96, 1996; and Huang, et al. J. Biol. Chem. 272:2927-2935, 1997).
Therefore, there is a need in the art to better study the HER-2/neu receptor and to determine if there are better regions of this protein available for using as a more sensitive diagnostic and prognostic indicator for breast cancer. Moreover, there is no procedure available to monitor for staging and prognosis of various adenocarcinomas, such as breast cancers, other than physically investigating adjacent tissue, such as regional lymph nodes and then sectioning the tissue by difficult histological techniques. Therefore, there is a need in the art to provide improved means for determining adenocarcinoma staging and further determining prognostic factors to guide appropriate treatment strategies. The present invention was made to address the foregoing needs in the art.
The present invention is based upon the initial identification of an N-terminally truncated HER-2/neu product. This product is approximately a 95 kDa polypeptide having in vitro kinase activity. Moreover, immunoprecipitation using domain specific antibodies was able to isolate this specific polypeptide from intracellular fragments for use as a diagnostic and prognostic indicator of various carcinomas without the severe dilution effects encountered by measuring ECD in blood/serum. The carcinomas for which the 95 kDa polypeptide will have diagnostic and prognostic value include, for example, carcinomas that overexpress HER-2, including breast, gastric, cervical, non-small cell lung, and prostate carcinomas.
The present invention provides a method for diagnostic and prognostic screening of a metastatic stage carcinoma that overexpresses HER-2, comprising:
(a) providing a suspected tissue sample having cells;
(b) lysing the cells to expose intracellular contents and form a lysate; and
(c) measuring the lysate for the presence of 95HER-2 polypeptide.
Preferably, the lysing step is followed by an additional step separating soluble from insoluble material of the lysate to remove dense fibrous material. Preferably, the measuring step utilizes an assay procedure selected from the group consisting of Western blotting, immunochemistry, ELISA, and combinations thereof. Preferably, the carcinoma that overexpresses HER-2 is selected from the group consisting of breast cancer, gastric carcinoma, prostate cancer, non-small cell lung carcinoma, and ovarian carcinoma.
The present invention provides a method for diagnostic and prognostic screening of a metastatic stage carcinoma that overexpresses HER-2, comprising:
(a) providing a suspected tissue sample having cells;
(b) providing an antibody that binds to a stub region of HER-2, wherein the stub region is a polypeptide sequence of SEQ ID NO. 1 or a fragment thereof; and
(c) determining the percentage of cells that have an exposed extracellular stub region.
Preferably, the means for determining the percentage of cells having an exposed extracellular stub region utilizes an assay procedure, wherein the assay procedure is selected from the group consisting of Western blotting, immunochemistry, red cell agglutination, ELISA, affinity chromatography, and combinations thereof. Preferably, the carcinoma that overexpresses HER-2 is selected from the group consisting of breast carcinoma, gastric carcinoma, prostate cancer, non-small cell lung carcinoma, and ovarian cancer.
A method for predicting the therapeutic effectiveness to treat a carcinoma that overexpresses HER-2 with a therapeutic agent, wherein the therapeutic agent is a HER-2 binding ligand, comprising:
(a) providing a tumor tissue sample having tumor cells contained therein;
(b) providing an antibody that binds to a stub region of HER-2, wherein the stub region is a polypeptide sequence, of SEQ ID NO. 1 or a fragment thereof, and
(c) determining the percentage of cells that have an exposed extracellular stub region, wherein a high percentage of tumor cells binding to the antibody indicates that the cancer will likely be resistant to the therapeutic agent.
Preferably, the means for determining the percentage of cells having an exposed extracellular stub region utilizes an assay procedure, wherein the assay procedure is selected from the group consisting of Western blotting, immunochemistry, red cell agglutination, ELISA, affinity chromatography, and combinations thereof. Preferably, the carcinoma that overexpresses HER-2 is selected from the group consisting of breast carcinoma, gastric carcinoma, prostate cancer, non-small lung carcinoma, and ovarian cancer. Preferably, the therapeutic agent is a humanized monoclonal antibody that binds to the extracellular domain of HER-2 (Herceptin).
A method for treating HER-2/neu-positive carcinomas, comprising administering an effective amount of a hydroxamate compound. Preferably, the hydroxamate compound is TAPI.
The present invention provides a method for determining node status in breast cancer prognosis, comprising:
(a) providing a suspected tissue sample having cells;
(b) dividing the tissue sample for measuring both p95HER-2 intracellularly and p185HER-2;
(c) lysing the cells to expose intraycellular contents and form a lysate for p95HER-2 assay;
(d) measuring the tissue sample for p185HER-2; and
(e) measuring the lysate for the presence of 95HER-2 polypeptide, wherein tissue samples that were both p95HER-2 positive and rich with p185HER-2 predict lymph node or other metastasis.
Preferably, the lysing step is followed by an additional step separating soluble from insoluble material of the lysate to remove dense fibrous material. Preferably, the measuring step utilizes an assay procedure selected from the group consisting of Western blotting, immunochemistry, ELISA, and combinations thereof.